The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
In specific binding assays, such as, e.g. immunoassays, DNA hybridization assays, receptor-binding assays, and cellular binding assays, generally the analytes to be measured are present at very low concentrations. Therefore, various labeling compounds have been developed that allow the labeling reactant to be detected and quantified at high sensitivity. In immunoassays and DNA hybridization assays time-resolved luminescence spectroscopy using lanthanide chelates is well known (e.g. I. Hemmilä, T. Stålberg, and P. Mottram (eds.), “Bioanalytical Applications of Labelling Technologies”, Wallac, Turku, 1994 and D. Wild (eds), “The Immunoassay Handbook”, Nature Publishing Group, 2001). Stable photoluminescent (referred in the context of this specification simply as luminescent) lanthanide chelates also have other applications, e.g. fluorescence microscopy and cytometry. Therefore, a number of attempts have been made to develop new highly luminescent chelates suitable for those types of time-resolved fluorometric applications. These include e.g. stable chelates composed of derivatives of pyridine (U.S. Pat. No. 4,920,195; U.S. Pat. No. 4,801,77; U.S. Pat. No. 4,761,481; U.S. Pat. No. 5,571,897; U.S. Pat. No. 5,859,215; Latva, M., Takalo, H., Mukkala, V. M., Matachescu, C., Rodriquez-Ubis, J. C. and Kankare, J., 1997, J. Luminescence, 75, 149; Takalo, H., Hemmilä, I., Sutela, T. and Latva, M., 1996, Helv. Chim. Acta, 79, 789), bipyridines (U.S. Pat. No. 5,216,134), terpyridines (U.S. Pat. No. 4,859,777, U.S. Pat. No. 5,202,423, U.S. Pat. No. 5,324,825) or various phenolic compounds (U.S. Pat. No. 4,670,572, U.S. Pat. No. 4,794,191, IT 42508A/89) as energy mediating groups and polycarboxylic acids as chelating parts. In addition, various dicarboxylate derivatives (U.S. Pat. No. 5,032,677, U.S. Pat. No. 5,055,578, U.S. Pat. No. 4,772,563), macrocyclic cryptates (U.S. Pat. No. 4,927,923, PCT WO 93/5049, EP 0 493 745), calixarenes (Sato, N. and Shinkai, S., 1993, J. Chem. Soc. Perkin Trans. 2, 621; Steemers, F. J., Verboom, W., Reinboudt, D. N., van der Tol, E. B. and Verhoeven, J. W., 1995, J. Am. Chem. Soc., 117, 9408), DTPA carbostril 124 conjugate (Selvin, P. R., Rana, T. M. and Hearst, J. E., 1994, J. Am. Chem. Soc., 116, 6029) and macrocyclic Schiff bases (EP 0 369 000) have been disclosed in patent applications and/or patents.
It is known that the luminescence lanthanide chelates are quenched in an aqueous solution. When water molecules are coordinated in the inner sphere of chelates, quenching is a result of an efficient, radiationless decay process involving vibronic coupling of lanthanide excited state and OH oscillation. The process is additive in regard to the number of OH oscillators, and hence the luminescence decay is inversely related to the number of bound water molecules. Various systems have been developed to avoid this phenomenon, such as using detergents and synergistic compounds, using high concentration of fluorine ions, removing water by drying prior to measurement, using a polymetric matrix, or measuring the luminescence in an organic solvent or in deuterium oxide. An ideal way to avoid direct aqueous quenching is to use stable, preferable nine dentate chelating agents, which do not allow the coordination of water with the chelated ion. In the above-mentioned chelates the lanthanide ion is normally coordinated to 7, 8 or 9 heteroatoms forming a seven-, eight- or nine-dentate chelate, respectively. Seven- and eight-dentate chelates contain from two to one water molecules and thus suffer aqueous quenching. It's generally assumed, that additional coordination atoms in nine—dentate chelates—having no water molecule in the first coordination sphere—don't have any additional positive effect in relation to aqueous quenching.
During an energy transfer process from an excited ligand to a lanthanide ion the energy undergoes intersystem crossing to one of ligand triplet states. The next step is a spin-forbidden transition of the energy, causing ligand phosphorescence, or an intra-molecular energy transfer to the lanthanide ion. Thermal decay, such as e.g. molecule thermal movement and rotation, is a known non-radiative deactivation process of mentioned triplet state. The lanthanide label chelates normally contains one reactive functional group for coupling the label to a biospecific binding reactant. Thus, in a labeled biomolecule the label may rotate and non-radiative deactivation of ligand triplet state is a possible phenomenon.
The general view is that several reactive binding groups in a label molecule cause cross reaction and formation of the biospecific binding reactant aggregates during the labeling process, and thus produce purification problems and decreased yield of labeled material.